Study On Mechanism Of High Nitrogen Input Promoting Outbreak Of Tobacco Bacterial Wilt

Ralstonia solanacearum is a soil-borne Gram-negative plant pathogenic bacterium that causes destructive bacterial wilt disease on many crops.R.solanacearum is widely distributed in tropical,subtropical and temperate regions,and is currently ranked as one of the ten most destructive bacterial pathogens worldwide due to its lethality,unusually wide host range,persistence and broad geographical distribution.R.solanacearum can survive in soil for a long time and generally invades host plants through natural root openings or root wounds.Once invade xylem vessels,it proliferates extensively and produces a huge number of exopolysaccharides（EPS）to block sap flow,resulting in quick stunting and wilting.Nitrogen,phosphorus and potassium are essential nutrients for plant growth,while their excessive inputs could usually lead to outbreak of bacterial wilt.We thus focused on effect of different input levels of nitrogen,phosphorus and potassium on development of bacterial wilt in tobacco plants,to reveal mechanism of outbreak of bacterial wilt under different nitrogen input levels.Main results are showing as follows:（1）Response of R.solanacearum to different concentrations of nitrogen and phosphorus based on minimal medium:Conditions of N₀ and P₀ severely inhibited growth of R.solanacearum in minimal medium,OD₆₀₀ of which could get to only about 0.1 at 48 hours post inoculation（hpi）.Condition of N_0.25 enable R.solanacearum to grow to an OD₆₀₀ as 0.84 at 48 hpi,which was significantly lower than those grown in conditions of N_0.75,N_1.0,N_1.25 and N_1.5.5 with OD₆₀₀ as 1.3.Conditions of P_0.25 and P_0.5.5 ensure R.solanacearum to grow to the maximum OD₆₀₀as 1.2 at 48 hpi,while the maximum OD₆₀₀ was slightly decreased with increased conditions of phosphorus.The type III secretion system（T3SS）plays an important role in process of R.solanacearum invading host root from soil.Concentrations of nitrogen and phosphorus in this minimal medium could also greatly affect expression levels of the T3SS,which was diminished in condition of N₀but substantially reduced in condition of P₀.It was greatly induced in condition of N_0.5,which was about two-older higher than that in the normal condition of N₁P₁,while it was slightly decreased with increased conditions of nitrogen.The T3SS expression was induced to the highest level in condition of P_1.0,which was slightly decreased with increased conditions of phosphorus.（2）Effect of different input levels of nitrogen,phosphorus and potassium on development of bacterial wilt disease in potted tobacco plants with the“3414”experiment and the effect of inoculation methods under different levels of nitrogen on the occurrence of tobacco bacterial wilt:“3414”test confirmed that high nitrogen treatment（N₃P₂K₂）induced tobacco bacterial wilt,and all tobacco died of the disease.Disease development was not affected with different input levels of nitrogen when tobacco plants were inoculated with the leaf-infiltration,which enables direct invasion into intercellular spaces of tobacco leaves,while it was significantly promoted with increased input levels of nitrogen when tobacco plants were inoculated with soil-soaking,which mimics natural invasion through roots.Note that only 6%of tobacco plants were wilted if without nitrogen input,while about 90%of tobacco plants could be wilted if with input level of the N₃.（3）Effects of different input levels of nitrogen on development of tobacco plants:nitrogen input significantly promoted growth and development of tobacco plants,including fresh weight of the above ground and plant height,but significantly reduced root length compared to that with less nitrogen input.R.solanacearum severely inhibited growth and development of tobacco plants that decreased the fresh weight of toots and plant height,but no effect on formation of root hairs which as significantly decreased in Arabidopsis thaliana plants when inoculated by R.solanacearum.In addition,R.solanacearum severely destroyed cortex structure of tobacco roots especially at highest input level of nitrogen（N₃）,which was 16,5.1 and 2.1-folder higher than those with nitrogen input of N₁ and N₂,respectively.（4）Effects of excessive input of nitrogen on growth of R.solanacearum in bulk soil and tobacco rhizosphere:growth of R.solanacearum in bulk soil and tobacco rhizosphere was significantly affected by excessive nitrogen input,i.e.,the N₃ input significantly increased cells number of R.solanacearum（Colony formation number,CFU）in bulk soil and tobacco rhizosphere at 5 days post inoculation（dpi）with soil-soaking inoculation,which were about 8.1-and 3.7-folder higher than those without nitrogen input（N₀）.The T3SS expression of R.solanacearum in soil at N₃ was significantly higher than N₀ at 1 and 3 days post inoculation;but the T3SS expression was slightly induced in nitrogen levels at 5dpi.All taken together,excessive nitrogen input is more beneficial for R.solanacearum to colonize in tobacco rhizosphere and destroy cortex structure of tobacco roots by causing cells death in root cortex,resulting efficient invasion in tobacco roots,and hence promotes outbreak of tobacco bacterial wilt.